1. Field of the Invention
This invention relates to covalently linked polypeptide cell modulators, each of which acts through a different and specific cell receptor to initiate complementary biological activities. Polypeptide cell modulators include lymphokines, monokines, interferons, polypeptide hormones or cytotoxins as well as modifications and active segments of such peptides. Also described are DNA sequences, plasmids and hosts capable of expressing the linked polypeptide cell modulators.
2. Description of Prior Art
One class of polypeptide cell modulators can be defined whose members exert an antiproliferative effect almost specifically on tumour cells and possess immunomodulatory activity, but lack antiviral activity. Among the members of this class are human lymphotoxin and tumour necrosis factor (Gray, P. W. et al. Nature 312, 721, 1984; Pennica D. et al. Nature 312, 724, 1984).
Human lymphotoxin (hLT) is a cytotoxin induced in lymphocytes by a specific antigen or by bacteria or parasites and has a cytotoxic or cytostatic action on a variety of tumour cells in vivo or in vitro. hLT has been implicated to play a role in cell-mediated immunity and its potent anti-tumour effect suggests it may be of value therapeutically (Ruddle, N. H. et al. Lymphokine Res. 2, 23, 1983).
Another class of lymphokine can be defined whose members induce an antiviral state in responsive cells, and also have antiproliferative and immunomodulating activity. Among the members of this class are leukocyte interferon (IFN-alpha), fibroblast interferon (IFN-beta) and immune interferon (IFN-gamma).
It has been reported that mixtures of type I interferons (IFN-beta or IFN-alpha) and type II interferons (IFN-gamma) are highly synergistic in exerting an antiviral or antiproliferative effect. (Fleishmann, W. R. et al. Infect. Immun. 26, 248, 1979; Czarniecki, C. W. et al. J. Virol. 49, 490, 1984).
In mixtures, much lower concentrations of type I and type II interferons can achieve a particular level of response. Several authors have also described IFN-gamma/hLT and IFN-alpha/hLT synergy or related synergies (Lee, S. H. et al. J. Immunol. 133, 1083, 1984; Stone-wolff, D. S. et al. J. Exp. Med. 159, 828, 1984; Williams, T. W. Lymphokine Res. 3, 113, 1984), European Patent Application (EPO 107 498), (EPO 128009).
However, in these instances, there was no disclosure of covalent linkage of the two classes of molecules that were synergistic.
Additional patent publications have described the primary amino acid sequences of human IFN-gamma (GB 2 107 718 A), the IFN-gamma (IFN X918) described herein (PCT 83/04053), IFN-alphas (U.S. Pat. No. 4,414,150- Aug. 11, 1983) and IFN-beta (e.g. GB 0689 70B; GB 2098996A). A modified IFN-beta (IFN X430) described herein is identical to human fibroblast IFN-beta except that amino acids 36 to 48 inclusive are replaced with amino acids 34 to 46 inclusive from human IFN-alpha 1 (European Patent Application 85105914.7 and (Taniguchi, T. at al. Nature 285, 547, 1980).
This invention encompasses mixed function proteins formed from covalently linked polypeptide cell modulators, each of which acts through a different and specific cell receptor to initiate complementary biological activities. Novel compounds of this invention are represented by the formula
R1xe2x80x94Lxe2x80x94R2
where R1 is a polypeptide cell modulator with one activity, R2 is a polypeptide cell modulator with a different but complementary activity. By complementary activity is meant activity which enhances or changes the response to another cell modulator. The polypeptide cell modulators are either directly bonded to one another or are each bound to a polypeptide linker segment. Thus L represents a chemical bond or a polypeptide linker segment to which both R1 and R2 are bound, most commonly L is a linear peptide to which R1 and R2 are bound by amide bonds linking the carboxy terminus of R1 to the amino terminus of L and the carboxy terminus of L to the amino terminus of R2. The linking group is generally a polypeptide of between 1 and 500 amino acids in length.
The term polypeptide cell modulator encompasses a large variety of peptides which elicit a biological response by binding to a specific binding site on a cell. It is known that mixtures of polypeptide cell modulators such as beta and gamma interferon exhibit a synergistic effect. In this invention the polypeptide cell modulators are bound together to produce the same synergistic effect as a mixture of the polypeptide cell modulators or a further enhanced effect or a different effect with the advantage of a single dosage form.
Compounds of this invention are preferably made by genetic engineering techniques. Thus genetic material (DNA) coding for one polypeptide cell regulator, peptide linker segment and the other polypeptide cell regulator is inserted into a suitable vector which is used to transform bacteria, yeast or mammalian cells. The transformed organism is grown and the protein isolated by standard techniques. The resulting product is therefore a new protein which has two complementary cell regulatory regions joined by a peptide linker segment as shown in the formula R1xe2x80x94Lxe2x80x94R2, wherein R1 and R2 represent polypeptide cell regulator regions and L represents the peptide linker segment.